The long-term goal of this project is to develop an understanding of the cellular and molecular mechanisms of human cytomegalovirus (HCMV) persistence in the host. HCMV is a species-specific virus that establishes a persistent/latent infection in the host after primary infection. Herpesviruses achieve latency/persistence by restricting expression of viral genes, thereby reducing acute replication while maintaining the ability to reactivate at a later stage. This process especially in CMV is not well understood but is likely to involve both cellular and viral factors. The recent discovery of RNA interference and the widespread expression of microRNAs has uncovered a new layer of post-transcriptional gene regulation that was previously unknown. Studies by our own group and others have identified over 70 miRNA genes encoded by multiple DNA viruses, the majority of which have been identified within the herpesvirus family. Although relatively little is known about the function of virally encoded miRNAs their potential ability to regulate multiple transcripts and the lack of an immunogenic response make miRNAs ideal candidates for the promotion and maintenance of a persistent or latent viral gene expression profile. Furthermore, preliminary data presented in this proposal suggests a possible role for at least one of the HCMV miRNAs in restricting acute replication of the virus. Following bioinformatics studies we have successfully identified a number of viral target transcripts of the HCMV miRNA UL112-1. These targets include the uracil DNA glycosylase gene, which resides directly antisense to UL112-1 and is directly cleaved by the miRNA, and the major transactivating protein IE72, which is regulated post-trancsriptionally via a target sequence within the 3'UTR of the messenger RNA. In this proposal we will extend the characterization of HCMV UL112-1 during acute infection and during persistent infection. In addition we will identify and characterize the HCMV gene targets of the other virally encoded miRNAs and analyze their contribution to regulation of the virus during acute infection. Lastly we will examine the expression and function of the HCMV miRNAs in monocyte macrophages and persistently infected endothelial cells. Elucidating the mechanisms of miRNA regulation of HCMV gene expression will provide an important contribution to herpesviruses and miRNA field as a whole.